Composition for prevention or treatment of bladder cancer

Abstract

The present invention relates to a composition for the prevention or treatment of bladder cancer, and particularly, to a composition for the prevention or treatment of cancer, comprising a nucleic acid that encodes proenkephalin (PENK) protein, which is a cancer inhibitory protein inhibited by methylation in cancer cells, in particular, PENK protein inhibited by methylation in bladder cancer cells. Thus, the composition inhibits the proliferation of bladder cancer cells.

Description

Composition for preventing or treating bladder cancer The present invention relates to a composition for preventing or treating bladder cancer, and more particularly, to a composition for preventing or treating bladder cancer that suppresses bladder cancer cell proliferation, comprising a nucleic acid encoding PENK (Proenkephalin) protein, a tumor suppressor protein that is suppressed by methylation in cancer cells, particularly PENK protein that is suppressed by methylation in bladder cancer cells. Gene therapy has been developed as a treatment to treat or prevent diseases by adding, modifying, or deleting new information to existing genes and delivering the genes to the patient's cells in the form of plasmid DNA (pDNA) or messenger RNA (mRNA) (Dunbar, C. E et al., Science 2018 1:12). The goal of gene therapy is to correct genetic defects in people with inherited or rare diseases and cure them completely. Recent technological innovations and R&D investments have led to a global gene therapy market growing at an average annual rate of 10%. Gene therapy is emerging as a blue chip in the new pharmaceutical market, and among them, mRNA is emerging as a promising therapeutic tool in vaccine development and protein replacement therapy. The principle of gene therapy is to introduce genes into cells in pDNA or mRNA form using viruses, liposomes, or antisense technology, thereby enabling them to function as therapeutic agents (Dunbar, C. E et al., Science 2018 1:12). mRNA therapeutics offer advantages over DNA or viral therapeutics in terms of safety, efficacy, and productivity. First, because mRNA is produced in the cytoplasm, the risk of mutations resulting from infection or genomic DNA insertion is reduced. Second, because mRNA undergoes various modifications within the cytoplasm, its half-life can be adjusted or the amount of translated protein can be increased, thereby enhancing intracellular stability. Third, mRNA is readily adaptable to in vitro experiments, allowing for rapid development and GMP production, and facilitates mass production (Wang, Y., Su et al. Molecular Therapy 2013 358-367). mRNA is RNA that transfers genetic information from DNA to ribosomes, leading to protein expression through mRNA translation. Recombinant mRNA, used for the development of therapeutics or vaccines, is produced from linear DNA using promoters (including but not limited to T7 and SP6) and RNA polymerase. This is followed by in vitro transcription, where 5' capping and poly A adenylation occur. This mRNA, similar to mature mRNA in the cytoplasm, consists of a 5' capping, 5' untranslated region (UTR), 3' UTR, poly A, and the target gene to be expressed. The untranslated region (5' or 3' untranslated region) influences mRNA stability and translational activation, increasing half-life and expression levels, depending on its sequence. Poly A also prevents mRNA degradation in the cytoplasm, thereby increasing stability and expression levels. Therefore, in order for mRNA therapeutics to efficiently and stably express proteins in the body, it is important to find the optimal non-translated region sequence and poly A length (Pardi, N et al., Nature reviews 2018 261-279). Meanwhile, extensive research is being conducted to treat cancer, the leading cause of death among humans. Currently, known cancer treatment methods include surgery, radiotherapy, chemotherapy, immunotherapy, and gene therapy. Among these, chemotherapy has severe side effects, radiotherapy is expensive and time-consuming, and exposes users to the risks of isotopes. Furthermore, immunotherapy suffers from low efficacy. Therefore, gene therapy, a novel treatment method that selectively introduces and expresses genes or proteins for cancer treatment into cancer cells and tissues, is emerging as a promising tool in the field of alternative treatment. The principle of gene therapy is to introduce genes into cells in the form of pDNA, PCR products, or mRNA using viruses, liposomes, or antisense technology, thereby enabling them to function as therapeutic agents (Dunbar, C. E et al., Science 2018 1:12). Among these, mRNA therapy offers advantages over DNA or viral therapies in terms of safety, efficacy, and productivity. Epigenetics refers to the phenomenon in which functional changes, such as gene expression, occur without changes in the DNA sequence, such as DNA methylation, covalent modifications of histone proteins, and ATP-dependent chromatin changes. A representative example is DNA methylation, which affects the expression of multiple genes by attaching a methyl group to the cytosine residue of CpG dinucleotides. Increased or decreased DNA methylation is primarily observed in cancer cells or tumor tissues, and active research on DNA methylation of specific genes in specific cancers is underway. Therefore, by regulating gene expression through DNA methylation and elucidating the mechanisms of expression regulation of tumor suppressor genes and oncogenes, it is expected to be useful not only for early diagnosis of cancer but also for prediction and personalized treatment (Kiselev IS et al., Acta Naturae. 2021). Under this technical background, the inventors of the present application discovered that the 5' expression control region of the PENK gene is specifically methylated in bladder cancer cells, thereby suppressing gene expression, and confirmed that when a nucleic acid encoding the PENK protein is treated in a cell line to cause overexpression, it exhibits an effect of suppressing the proliferation of bladder cancer cell lines, thereby completing the present invention. Summary of the invention The purpose of the present invention is to provide a composition, method or use for preventing or treating bladder cancer. To achieve the above purpose, the present invention provides a composition for preventing or treating bladder cancer comprising a nucleic acid encoding PENK (Proenkephalin) protein. The present invention also provides a method for preventing or treating bladder cancer, comprising administering to a subject a nucleic acid encoding a PENK (Proenkephalin) protein. The present invention further provides a use of a nucleic acid encoding a PENK (Proenkephalin) protein for the preparation of a composition for preventing or treating bladder cancer. The present invention provides a composition for preventing or treating bladder cancer, comprising a nucleic acid encoding PENK (Proenkephalin) protein and a carrier. The present invention also provides a method for preventing or treating bladder cancer, comprising administering to a subject a nucleic acid encoding a PENK (Proenkephalin) protein and a carrier. The present invention further provides a use of a nucleic acid encoding a PENK (Proenkephalin) protein and a carrier for preparing a composition for preventing or treating bladder cancer. Figure 1 shows the methylation of the promoter region of the gene encoding proenkephalin (PENK) and the mRNA and protein expression. ((A): Measurement of methylation of the PENK gene promoter region using pyrosequencing in normal cell lines and bladder cancer cell lines; (B): Confirmation of mRNA expression of the PENK gene in the bladder cancer cell line (T24); (C): Confirmation of protein expression of the PENK gene in the bladder cancer cell line (T24)). Figure 2 illustrates the process of producing template DNA for mRNA expression of the PENK gene using the PCR method. Figure 3 shows the mRNA expression of the control and PENK genes confirmed using the IVT (In vitro transcription) method. Figure 4 shows the PENK gene mRNA produced in Figure 3 transfected into a bladder cancer cell line (T24), and the expression of PENK protein and apoptosis-inducing factors was confirmed through Western blotting. ((A): PENK protein expression confirmed; (B) Apoptosis-inducing factor in bladder cancer cells confirmed) Figure 5 shows the effect of PENK gene mRNA produced in Figure 3 on the growth and proliferation of bladder cancer cells (T24) transfected with the cell line. ((A) Cell proliferation inhibition by PENK mRNA observed under a microscope; (B) The image is digitized and represented as a graph.) Figure 6 shows the effect of transfecting the PENK gene mRNA produced in Figure 3 into a bladder cancer cell line (T24) on the formation of bladder cancer cells. ((A) Observation of the inhibition of cell cluster formation by PENK mRNA through photography; (B) The image is digitized and represented as a graph.) Figure 7 shows the effect of PENK gene mRNA produced in Figure 3 on the wound healing ability of bladder cancer cells (T24) transfected with the cell line. ((A) Microscopic observation of whether PENK mRNA inhibits cell wound healing; (B) Graphical representation of the image converted to numbers.) Figure 8 shows the effect of PENK gene mRNA produced in Figure 3 on the invasion ability of bladder cancer cells (T24) transfected with the cell line. ((A) Microscopic observation of whether cell invasion ability was inhibited by PENK mRNA; (B) The image is digitized and represented as a graph.) Detailed description and specific implementation examples of the invention Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art. In previous studies, we have reported that the PENK gene is methylated in bladder cancer (Oh et al., BMC cancer 2022; Oh et al., J Mol Diagn 2023). Furthermore, we demonstrated that PENK methylation is a biomarker for the early diagnosis of bladder cancer using urine, and that it can be used to diagnose bladder cancer early in patients with hematuria suspected of having the disease. In a specific embodiment according to the present invention, it was discovered that the 5' expression control region of the PENK gene was specifically methylated in bladder cancer cells, thereby suppressing gene expression, and it was confirmed that when a PENK mRNA-based complex was treated to a bladder cancer cell line and overexpressed, it exhibited an effect of suppressing the proliferation of the bladder cancer cell line. Based on this, from one aspect, the present invention relates to a composition for preventing or treating bladder cancer, comprising a nucleic acid encoding the PENK (Proenkephalin) protein. The present invention also relates to a method for preventing or treating bladder cancer, comprising administering to a subject a nucleic acid encoding the PENK (Proenkephalin) protein. Furthermore, the present invention relates to the use of a nucleic acid encoding the PENK (Proenkephalin) protein in the manufacture of a composition for preventing or treating bladder cancer. Nucleic acids are not limited to those containing nucleotides as their basic structural units, but may include, for example, RNA or DNA. In the case of RNA, it is synthesized from ribonucleotides, but in a sample containing RNA in which some of the nucleosides introduced during the synthesis process are modified, it can be a gene that exhibits a desired function in an organism, as RNA that is substantially free of errors or damage. 'DNA' means a polynucleotide containing the sugar deoxyribose and composed of the purine or pyrimidine bases adenine, thymine, cytosine or guanine. 'RNA' refers to a polynucleotide containing a ribose sugar, typically uracil rather than thymine as one of the pyrimidine bases. RNA may comprise a single-stranded molecule transcribed from DNA, and has a linear sequence of nucleotide bases complementary to the transcribed DNA strand. 'Nucleotide' means a glycoside comprising a sugar moiety, a base moiety, and a covalently linked linkage group, such as a phosphate or phosphorothioate internucleotide linkage group. It may include both naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides, optionally including modified sugar and / or base moieties. 'Ribonucleotide' or 'deoxyribonucleotide' can include both natural and synthetic, unmodified and modified ribonucleotides or deoxyribonucleotides. Modifications can include changes to the sugar moiety, base moiety, and / or linkages between ribonucleotides or deoxyribonucleotides within the oligonucleotide. In relation to the above nucleic acids, the terms "polynucleotide," "nucleotide," "nucleotide sequence," and "oligonucleotide" are used interchangeably. Polynucleotides of any length may comprise polymeric forms of nucleotides, deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. Polynucleotides may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure may occur before or after assembly of the polymer. "Polynucleotide" means a polymeric form of nucleotides comprising nucleotides of any length, including deoxyribonucleotides and / or ribonucleotides, or their analogs. A polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. The structure of a polynucleotide may be considered with reference to the 5' or 3' termini, which indicate the directionality of the polynucleotide. Adjacent nucleotides in a single polynucleotide strand are typically linked by phosphodiester bonds between the 3' and 5' carbons. However, other intranucleotide linkages, such as those involving methylene or phosphoramidate bonds, may also be used. This means that each of the 5' and 3' carbons may be exposed at either end of the polynucleotide, which may be referred to as the 5' and 3' termini or termini. The 5' and 3' termini may also be referred to as the phosphoryl (PO4) and hydroxyl (OH) termini, respectively, due to the chemical groups attached to them. The polynucleotide may be used interchangeably with nucleic acids and may include both double-stranded and single-stranded molecules. Examples of polynucleotides include, but are not limited to: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), genomic DNA, a genomic DNA fragment, an exon, an intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, an isolated DNA of any sequence, an isolated RNA of any sequence, a nucleic acid probe, a primer, or an amplified copy of any of the above. The polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs (including nucleotides having unnatural bases, nucleotides having modified natural bases such as aza- or deza-purines, etc.). The polynucleotide may be composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T). Uracil (U) may exist as a natural substitute for thymine, for example, when the polynucleotide is RNA. Uracil can also be used in DNA. Therefore, the term "sequence" refers to an alphabetical representation of a polynucleotide or any nucleic acid molecule, including both natural and unnatural bases. The above nucleic acid may include a sequence represented by SEQ ID NO: 1 or a sequence having at least 90% homology thereto. [Sequence number 1] The above homology means a sequence that exhibits at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, or 99% or more homology when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequences are analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLAST is accessible at www.ncbi.nlm.nih.gov / BLAST / . A method for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_ help.html. Based on this, the nucleic acid sequence of the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology compared to the specified sequence or the entire sequence described in the specification. The nucleic acid of the present invention may be mRNA. The mRNA according to the present invention may comprise the sequence of SEQ ID NO: 13. [Sequence number 13] mRNA can be synthesized using any of a variety of known methods. For example, mRNA according to the present invention can be synthesized via in vitro transcription (IVT). IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. Conditions may vary depending on the application. The mRNA according to the present invention can be purified so as to be free from the presence of reagents, i.e., impurities. In some embodiments, the present invention can be used to purify mRNA containing one or more modifications, typically to enhance stability. In some embodiments, the one or more modifications are selected from modified nucleotides, modified sugar phosphate backbones, and 5' and / or 3' untranslated regions. In some embodiments, the present invention can be used to purify unmodified in vitro-synthesized mRNA. mRNA can be modified to enhance its stability. Modifications of mRNA can include, for example, modifications to the nucleotides of the RNA. Accordingly, modified mRNA according to the present invention can include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, the antibody encoding mRNA (e.g., heavy and light chain encoding mRNA) comprises a purine (adenine (A), guanine (G)) or a pyrimidine (thymine (T), cytosine (C), uracil (U)), and modified nucleotide analogues or derivatives of purines and pyrimidines, for example, 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-Methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 1-methyl-pseudouracil, queosine, β-D-mannosyl-queosine, wybutoxosine, and naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides) including but not limited to phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine. In one embodiment, the nucleic acid comprises a 5' untranslated region (UTR) located upstream of the nucleic acid encoding the PENK protein; and a 3' untranslated region (UTR) located downstream of the coding region, wherein the 5' untranslated region (UTR) can be included in a genetic construct comprising a leader sequence of a coronavirus. The SARS-CoV-2 leader sequence and intergenic sequence that can act as 5' UTR can be connected, and the coding sequence of the gene to be expressed immediately thereafter, i.e., the nucleic acid encoding the PENK protein and the 3' UTR sequence and polyA sequence of SARS-CoV-2 can be connected to introduce the gene into a cell. At this time, in order to express the mRNA of the desired PENK protein coding gene in vitro, a promoter sequence (T7, SP7, etc.) can be linked to the 5' end of the above sequence to produce the mRNA of the PENK protein coding gene using the IVT (in vitro transcription) method. Specifically, the present invention provides a genetic construct comprising a coding region for mRNA expression of a PENK protein coding gene; a 5' untranslated region (UTR) located upstream of the coding region; and a 3' untranslated region (UTR) located downstream of the coding region, wherein the 5' untranslated region (UTR) may comprise a genetic construct including a leader sequence of a coronavirus. Regarding the leader sequence, the genomic RNA and the subgenomic mRNAs that are transcribed and expressed among coronaviruses have a leader sequence of approximately 72 to 77 bp at the 5' end in common. This is a unique characteristic of coronaviruses and is the most abundant target among viral sequences in infected cells. This is because all subgenomic RNAs of coronaviruses exhibit a leader joining phenomenon, in which a leader sequence corresponding to approximately 72 bp derived from the 5' end of the genomic RNA binds to the 5' end of each subgenomic RNA. Therefore, the leader sequence has the highest copy number among viral genes in the cell, followed by the copy number of the subgenomic RNA encoding the N protein. RNA includes, for example, mRNA. RNA is usually an abbreviation for ribonucleic acid. It is a polymer made of nucleic acid molecules, i.e., nucleotides. Nucleotides are usually monomers of adenosine monophosphate, uridine monophosphate, guanosine monophosphate, and cytidine monophosphate, linked together by a so-called backbone. The backbone is formed first by a sugar, such as ribose, and second by phosphodiester bonds between adjacent monomers, the phosphate moieties. The specific sequence of monomers is called an RNA sequence. RNA can usually be obtained by transcription of a DNA sequence, for example, within a cell. In eukaryotic cells, transcription typically occurs in the nucleus or mitochondria. In the body, DNA transcription is usually processed into mRNA, messenger RNA. For example, RNA processing in eukaryotic cells involves various posttranscriptional modifications, such as splicing, 5'-capping, polyadenylation, export from the nucleus or mitochondria, and the like. Messenger RNA (MRNA) typically provides a nucleotide sequence that can be translated into the amino acid sequence of a specific peptide or protein. Typically, mRNA contains a 5'-cap, a 5'UTR, an open reading frame, a 3'UTR, and a poly(A) sequence. Aside from messenger RNA, several non-coding RNAs exist that can be involved in the regulation of transcription and / or translation. The 5'UTR is located 5' of the open reading frame. The 5'UTR begins at the transcription start site and ends at the nucleotide before the start codon in the open reading frame. The 5'UTR may contain elements that regulate gene expression, such as a ribosome binding site. The 5'UTR may be post-transcriptionally modified, for example, by the addition of a 5'-cap. The 5'UTR corresponds to the sequence of the mature mRNA located between the 5'cap and the start codon. Specifically, the 5' untranslated region (UTR) may include a sequence as SEQ ID NO: 2. [Sequence number 2] The 3'UTR is a portion of an mRNA typically located between the protein-coding region (i.e., open reading frame) and the poly(A) sequence. The 3'UTR of an mRNA is not translated into an amino acid sequence. The 3'UTR sequence is typically encoded by a gene, which is then transcribed into mRNA during gene expression. The genomic sequence is first transcribed into mRNA, which contains optional introns. The mRNA then undergoes steps such as 5' capping, splicing, and modification of the 3' end, such as polyadenylation of the 3' end, as well as optional endo- or exonuclease digestion. The 3'UTR is located immediately 3' to the stop codon in the protein-coding region and includes the nucleotides immediately 5' to the poly(A) sequence. The above 3' untranslated region (UTR) may include a 3' UTR sequence of SARS-CoV-2 and may include a sequence of SEQ ID NO: 3. [Sequence number 3] It can be a sequence of several nucleotide triplets that can be typically translated into a peptide or protein. The open reading frame preferably comprises a start codon, i.e., a combination of three subsequent nucleotides at its 5'-end, which typically encode the amino acid methionine (ATG or AUG), and a subsequent region that is usually a multiple of three nucleotides in length. The ORF is preferably terminated by a stop codon (e.g., TAA, TAG, TGA). This is the only stop codon in the open reading frame. Therefore, in the context of the present invention, an open reading frame is a nucleotide sequence consisting of a number of nucleotides that can be divided into three, preferably beginning with a start codon (e.g., ATG or AUG) and preferably ending with a stop codon (e.g., TAA, TGA, or TAG, or UAA, UAG, UGA, respectively). The open reading frame can be isolated or incorporated into a longer nucleic acid sequence, such as a vector or mRNA. An open reading frame may also be referred to as a "protein coding region." Specifically, according to the present invention, it comprises a nucleic acid encoding a PENK protein having a sequence represented by SEQ ID NO: 1. According to the present invention, a promoter and / or poly(A) sequence may be additionally included. The promoter may be located upstream of the 5' untranslated region (UTR). The promoter may include elements necessary for transcription, such as an RNA polymerase promoter. It may include a phage RNA polymerase promoter, such as SP6 or T7, preferably a T7 promoter encoding an mRNA sequence. The length of the poly(A) sequence can vary. For example, the poly(A) sequence can have a length of from about 20 adenine nucleotides to about 300 adenine nucleotides, preferably from about 40 to about 200 adenine nucleotides, more preferably from about 50 to about 100 adenine nucleotides, such as from about 60, 70, 80, 90, or 100 adenine nucleotides. Specifically, in an embodiment according to the present invention, the length of the poly(A) sequence may be 65 nucleotides in length. The poly(A) sequence may be located downstream of the 3' untranslated region (UTR). For example, the poly(A) sequence may be linked directly or via a linker, for example via a linker of 1-50, preferably 1-20, nucleotides, or via a stretch of nucleotides, such as 2, 4, 6, 8, 10, 20, etc. nucleotides. In a specific embodiment, the present invention may comprise a genetic construct comprising a sequence represented by SEQ ID NO: 4. [Sequence number 4] The composition may include a delivery means for delivering the nucleic acid, for example, mRNA expressed or synthesized from the nucleic acid, specifically an acceptable carrier. Based on this, the present invention relates to a composition for preventing or treating bladder cancer, comprising a nucleic acid encoding PENK (Proenkephalin) protein and a carrier. The present invention also relates to a method for preventing or treating bladder cancer, comprising administering to a subject a nucleic acid encoding a PENK (Proenkephalin) protein and a carrier. The present invention further relates to the use of a nucleic acid encoding a PENK (Proenkephalin) protein and a carrier for the manufacture of a composition for preventing or treating bladder cancer. The mRNA may be delivered via nanoparticles, for example, via gold nanoparticles. The above gold nanoparticles can have their surfaces modified. Specific examples of modifications are described in Acc Chem Res. June 18, 2019; 52(6): 1496-1506 and Pharmaceutics 2021, 13, 900, which may be incorporated herein by reference. The mRNA can be delivered to cells by linking gold nanoparticles to the mRNA and forming a complex with a cationic endosomal disruptive polymer (Nature Biomedical Engineering volume 1, pages 889-901 (2017)). The cationic endosomal disruptive polymer can be, for example, polyethylene imine, poly(arginine), poly(lysine), poly(histidine), poly-[2-{(2-aminoethyl)amino}-ethyl-aspartamide] (pAsp(DET)), a block copolymer of poly(ethylene glycol) (PEG) and poly(arginine), a block copolymer of PEG and poly(lysine), or a block copolymer of PEG and poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-pAsp(DET)). In some cases, the surface may use gold particles modified with arginine. Gold particles modified with arginine can be assembled with a nuclease or a polynucleotide encoding the same and / or a cleavage factor or a polynucleotide encoding the same, thereby fusing with the membrane of the target cell and translocating into the cytoplasm (ACS Nano. 2017, 11:2452-2458). The expressed mRNA can be delivered via liposomes, LNPs (lipid nano-particles), or various nanoparticles. Liposomes or LNPs contain cationic lipids, non-cationic lipids, or neutral lipids, and may additionally contain other lipids such as PEG (polyethylene glycol) or cholesterol. Such mRNA delivery systems are specifically described in U.S. Patent Publication Nos. 2018 / 0311176, 2019 / 0032051, and 2021 / 0046192, and International Patent Publication Nos. WO2018 / 081480, WO2020 / 097540, WO2020 / 097548, and WO2021 / 007278, which are incorporated herein by reference. The cationic lipid may be, for example, Lipofectamine. 상기 양이온성 지질은 예를 들어, 미국특허공개 제2018 / 0311176호, 제2019 / 0032051호 등을 통해 구체적으로 예시되어 있으며, 예를 들어 N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9, 12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1, 1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, β-L-arginyl-2, 3-L-diaminopropionic acid-N-palmityl-N-oleylamide trihydrochloride, N′,N′-dioctadecyl-N-4, 8-diaza-10-aminodecanoylglycine amide.

[0071] , 1,2-dilinoleyloxy-3-dimethylaminopropane, DLin-KC2-DMA, amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA, 1), 1,2-distearloxy- / V,N-dimethylaminopropane (DSDMA), dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), DLin-D-DMA, C12-200, 98N12-5, (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimemylhexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-8-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacos-18-en-10-amine, (17Z)-N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-10-amine, (15Z)-N,N-dimethyleptacos-15-en-10-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylhentriacont-22-en-10-amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimeth-yl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(1 S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, R-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propa-n-2-amine, S-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octy-loxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-Roctyloxy)methyl]ethyl}pyrro-lidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azet-idine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-ylo-xy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]pr-opan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-am-ine; (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(o-ctyloxy)propan-2-amine, (2 S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propa-n-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-di-methylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)pr-opan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpro-pan-2-amine, (2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amin-e,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-di-en-1-yloxy]propan-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]-methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-am-ine and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,2-trien-10-amine, 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”), dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE), DMRIE-HP, Lipofectamine (DOSPA), 3b-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Choi”), N-(1,2-dimyhstyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (“DMRIE”), 1,2-Dioleoyl-3-dimethylammonium-propane (“DODAP”), DMDMA, cationic lipid-based transfection reagents TransIT-TKO, LIPOFECTIN, Lipofectamine, OLIGOFECTAMINE or DHARMAFECT, DSDMA, DODMA, DLinDMA, DLenDMA, gamma-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3-DM A, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), DLin-M-C3-DMA (also known as MC3) or (DLin-MP-DMA)(also known as 1-B11), or mixtures thereof, but are not limited thereto. no., The above non-cationic lipid is specifically exemplified in U.S. Patent Publication Nos. 2018 / 0311176 and 2019 / 0032051, and may be, for example, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanolamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, or lysylphosphatidylglycerol.경우에 따라서, 상기 비양이온성 지질은 예를 들어, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG)일 수 있으나, 이에 제한되는 것은 아니다. The above neutral fat is specifically exemplified in U.S. Patent Publication Nos. 2018 / 0311176 and 2019 / 0032051, and may include, but is not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, or cerebrosides. PEG fat may be included to prevent aggregation of particles generated during the above mRNA delivery. PEG fat is specifically exemplified in U.S. Patent Publication Nos. 2018 / 0311176 and 2019 / 0032051, and examples thereof include PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. As a non-limiting example, PLGA may be conjugated to a lipid-terminating PEG forming PLGA-DSPE-PEG, PEG lipid is selected from PEG-c-DOMG and 1,2-Dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (PEG-DMG), 1,2-Distearoyl-sn-glycerol, methoxypolyethylene Glycol (PEG-DSG), PEG-c-DOMG, 1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG-DSG) 1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol (PEG-DPG), PEG-lipid conjugates such as, eg, PEG coupled to dialkyloxypropyls (eg, PEG-DAA conjugates), PEG coupled to diacylglycerols (eg, PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides, cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers (eg, ATTA-lipid conjugates), and mixtures thereof. In some embodiments, the PEG is a PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), a PEG-distearyloxypropyl (C18), PEG-c-DOMG, PEG-DMG, or a mixture thereof, but is not limited thereto. Peptides can be used for the above mRNA delivery. The peptide must have a cationic charge to electrostatically interact with the anionic phosphate group of the nucleic acid, and may include positively charged amino acids to electrostatically interact with the phosphate group. Specific details of peptides usable for mRNA delivery are described in AIMS Biophysics, 7(4): 323-338, which is incorporated herein by reference. Protamine may be included as a peptide usable for the above mRNA delivery. Protamine is a small, cationic, arginine-rich nuclear protein that contributes to the stability of DNA during spermatogenesis in the testis. Protamine stabilizes mRNA molecules, enabling efficient delivery. A protamine-mRNA complex is specifically described in U.S. Patent No. 9,352,028, which is incorporated herein by reference. Cell-penetrating peptides (CPPs) may also be promising cationic molecules for mRNA delivery. Amphipathic CPPs, such as the arginine-rich RALA peptide (WEARLARALARALARHLARALARALRACEA), RALA, LAH4 (KKALLALALHHLAHLALHLALALKKA), and LAH4-L1 (KKALLAHALHLLALLALHLAHALKKA), can be used to deliver mRNA molecules. In some cases, in addition to the liposome or LNP (lipid nanoparticle), a peptide may be additionally included for mRNA delivery. The peptide can provide nucleic acid packaging and protect DNA or RNA from degradation either intracellularly or extracellularly. Examples of such peptides are specifically described in U.S. Patent Publication No. 2021 / 0170046, which is incorporated herein by reference, but is not limited thereto. The composition may further comprise one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers must be compatible with the active ingredient of the present invention, and may include saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components mixed together. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, suspension, or emulsion. In particular, it is preferable to provide the composition in a lyophilized form. A method commonly known in the art to which the present invention pertains can be used for the preparation of a lyophilized formulation, and a stabilizer for lyophilization may be added. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the field or a method disclosed in Remington's pharmaceutical Science (Mack Publishing company, Easton PA). The content and administration method of the active ingredients, etc. included in the composition of the present invention can be determined by a person skilled in the art based on the symptoms and severity of the disease of a typical patient. Furthermore, the composition can be formulated in various forms, such as powders, tablets, capsules, liquids, injections, ointments, and syrups, and can also be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles. The composition of the present invention can be administered orally or parenterally. The route of administration of the composition according to the present invention is not limited to these, but for example, it can be administered intrabronchially, orally, intravenously, intramuscularly, intraarterially, intramedullaryly, intrathecally, intracardiacly, transdermally, subcutaneously, intraperitoneally, enterally, sublingually, or topically. The dosage of the composition according to the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, method, excretion rate, or disease severity, and can be easily determined by a person skilled in the art. In addition, the composition of the present invention can be formulated into a suitable dosage form using known techniques for clinical administration. The subject may be a subject suffering from bladder cancer. In addition, the subject may be a mammal, and preferably a human. “Treatment” refers to any sign of success in the treatment or amelioration of an impairment, pathology or condition, including any subjective or objective parameter, such as alleviation; remission; making symptoms or impairments more tolerable to the patient; reducing the pathology or condition; slowing the rate of regression or decline; creating a less debilitating final point of regression; or improving the physical or mental well-being of the patient. Treatment or amelioration of symptoms may be based on objective or subjective parameters, including physical examination, neuropsychiatric testing and / or psychiatric evaluation. An "effective amount" is generally an amount sufficient to reduce the severity or frequency of a symptom, eliminate a symptom, its underlying cause, prevent the onset of a symptom or its underlying cause, or ameliorate or correct impairment resulting from or associated with a disease state. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to correct a disease state or symptom, particularly a condition or symptom associated with a disease state, or otherwise prevent, impede, delay, or reverse the progression of a disease state or any other undesirable symptom associated with the disease in any way. A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, has the intended prophylactic effect, e.g., preventing or delaying the onset of a disease state, or reducing the likelihood of onset (or recurrence) of a disease state or its associated symptoms. Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples. Example 1. PENK expression in bladder cancer cell lines according to methylation. To confirm the correlation between targeted PENK protein expression and DNA methylation, we first examined the methylation level of the bladder cancer cell line T24 using pyrosequencing. As a result, we confirmed higher methylation in T24 compared to normal cells (Fig. 1 (A)). Subsequently, we confirmed mRNA and protein expression using RT-PCR and Western blot, but PENK was not detected (Fig. 1 (B), (C)). Therefore, we initially confirmed that high methylation suppressed mRNA and protein expression. Example 2. Construction of a PENK gene mRNA construct for PENK protein expression suppressed by methylation. 2-1. Production of IVT template for PENK mRNA expression The PENK mRNA construct for PENK protein expression was prepared based on the mRNA construct production method previously developed by the present inventors (International Patent Application Publication No. WO2023 / 063769). The IVT template sequence previously produced through this method was confirmed and presented in the table below. In addition, to produce an IVT template for PENK mRNA delivery, the PENK gene coding sequence was linked immediately after the 5' UTR of the N gene of SARS-CoV-2, followed by the SARS-CoV-2 3' UTR sequence and poly A sequence (65 nucleotides) using PCR (Fig. 2). To synthesize 5' UTR using 1 ng of plasmid DNA for control RNA expression produced above as a template, 10 pmoles of forward primer (SEQ ID NO: 5), 10 pmoles of reverse primer (SEQ ID NO: 6), and 10 μl of 2X pfu Master Mix (Biofact, Daejeon, South Korea) were added. To synthesize 3' UTR, 10 pmoles of forward primer (SEQ ID NO: 9), 10 pmoles of reverse primer (SEQ ID NO: 8), and 10 μl of 2X pfu Master Mix (Biofact, Daejeon, South Korea) were added. PCR (Applied Biosystem) reaction conditions were as follows: 95°C for 2 min (1 cycle); 95°C for 20 sec - 60°C for 40 sec - 72°C for 1 min (30 cycles); 72°C (5 min). The constructed PCR products were confirmed by electrophoresis on agarose gel (2%). The PENK gene was amplified using 10 pmoles of a forward primer (SEQ ID NO: 10) in which the 3'-terminal sequence of the 5' UTR and the 5'-terminal sequence of the PENK gene were linked using pCMV6-PENK1 ng containing the PENK gene as a template, 10 pmoles of a reverse primer (SEQ ID NO: 11) in which the 3'-terminal sequence of the PENK gene and the 5'-terminal sequence of the 3' UTR were linked, and 10 μl of 2X pfu Master Mix (Biofact, Daejeon, South Korea) was added. The PCR (Applied Biosystem) reaction conditions were as follows: 95℃ for 2 min (1 cycle); 95℃ for 20 sec - 60℃ for 40 sec - 72℃ for 2 min (30 cycles); 72℃ (5 min). The constructed PCR products were confirmed by electrophoresis on agarose gel (2%). A PCR method was used to link the T7 promoter sequence, 5' UTR sequence, PENK gene sequence, 3' UTR sequence, and 65 nucleotide poly A sequence.After mixing equal amounts of 100 pg of each PCR product prepared above, 10 pmoles of forward T7 promoter sequence primer (SEQ ID NO: 5) was used as a template, 10 pmoles of reverse primer (SEQ ID NO: 8) connecting 20 nucleotides of the 3' end of the 3' UTR sequence and a 65 nucleotide polyA sequence, and 10 μl of 2X pfu Master Mix (Biofact, Daejeon, South Korea) were added. PCR (Applied Biosystem) reaction conditions were as follows: 95°C for 2 min (1 cycle); 95°C for 20 sec - 60°C for 40 sec - 72°C for 2 min (30 cycles); 72°C (5 min). The prepared PCR products were confirmed by electrophoresis on agarose gel (2%). The PCR product was ligated into the pTOP Blunt V2 vector (Enzynomics, Daejeon, South Korea) and transformed into DH5α (Enzynomics, Daejeon, South Korea). After transformation, the cells were plated on solid medium (Bioloard, Daejeon, South Korea) containing ampicillin (50 μg / ml, Sigma Aldrich, USA) and cultured at 37°C for 16 h. Plasmid DNA was extracted using HiGeneTM Plasmid Mini Prep Kit (Ver. 2.0) (Biofact, Daejeon, South Korea), and the constructed IVT template sequence was confirmed by Sanger sequencing (SEQ ID NO: 4). [Sequence number 4] 2-2. Confirmation of Control mRNA and PENK mRNA structures Based on Example 2-1, to produce each mRNA, 10 pmole of the forward primer (SEQ ID NO: 12) corresponding to the front sequence of the T7 promoter, 10 pmole of the reverse primer (SEQ ID NO: 8) linked to the 3' UTR and polyA 65 of nucleotide, and 10 μl of 2X pfu Master Mix (Biofact, Daejeon, South Korea) were added to perform PCR in a total volume of 20 μL. The PCR reaction conditions were as follows: 95°C for 2 min (1 cycle); 95°C for 20 sec - 60°C for 40 sec - 72°C for 2 min 30 sec (30 cycles); 72°C (5 min). The amplified PCR products were purified using a Qiaquick® Gel Extraction Kit (QIAGEN, Hilden, Germany) after electrophoresis on a 2% agarose gel and used as templates for IVT. IVT was performed using the Hiscribe T7 ARCA mRNA kit (NEB) according to the manufacturer's instructions. 1 μg of the above-mentioned PCR product was used as a template, and 10 μl of 2X ARCA / NTP MIX and 2 μl of T7 polymerase were added and incubated at 37°C for 16 h to allow mRNA synthesis and the addition of anti-reverse cap analog (ARCA) to the 5' end. After the reaction, 2 μl of DNase I was added and incubated at 37°C for 15 min to remove the DNA template. After the IVT reaction, the reaction solution was purified using the Monarch RNA Cleanup kit (NEB, Massachusetts, USA) and eluted in 50 μl of RNase-free distilled water. The purified RNA was quantified using nanodorp and the mRNA product was confirmed by electrophoresis on an agarose gel (1%, 0.5X TBE) (Fig. 3). After checking the size after electrophoresis, it was confirmed that it matched the expected size. Example 3. Confirmation of Control and PENK protein expression in human cell lines 3-1. Transfection and cell harvesting Bladder cancer T24 cell line was seeded at 0.8 x 10 in a 60Ø plate (SPL, Pocheon, Korea). 6 After spreading the cells per well, they were cultured in a 37℃ incubator under 5% CO2 conditions for 24 hours. mRNA synthesized by the IVT method and Lipofectamine Messenger MAX were mixed (1:1.5, w:v) and transfected into T24 cells. After culturing for 24 hours in a 37℃ incubator under 5% CO2 conditions, the cells were harvested. Proteins were extracted from the harvested cells using pro-prep (iNtRON biotechnology, Seongnam, South Korea) according to the manufacturer's instructions. 3-2. Confirmation of PENK protein expression and PENK apoptosis function through Western blot The extracted proteins were subjected to SDS-PAGE using a Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-rad, California, USA) and then transferred to a nitrocellulose membrane (Bio-rad, California, USA). The proteins transferred to the membrane were blocked with 5% skim milk (Bio-rad, Seoul, South Korea) for 1 hour, and then PENK polyclonal antibody (Invitrogen, Massachusetts, USA) diluted in 5% skim milk was added to the membrane, which was then incubated at 4°C for 16 hours. After washing three times with 1X TBST for 10 minutes, the secondary antibody, mouse anti-rabbit IgG HRP (Santa Cruz Biotechnology, Texas, USA), diluted in 5% skim milk was added, and incubated at room temperature for 2 hours, followed by washing three times with 1X TBST for 10 minutes. Luminol / enhancer solution (Cyanagen, Bologna, Italy) and Peroxide solution (Cyanagen, Bologna, Italy) were mixed in a 1:1 ratio and applied to the membrane. After reacting for 1 minute at room temperature, protein expression was confirmed using fusion solo X (Vilber, Eberhardzell, Germany) equipment (Figure 4 (A)). To confirm the cell death function of PENK, the protein expression of the cell death factors PARP and Caspase-3 was also confirmed. The extracted proteins were subjected to SDS-PAGE using a Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-rad, California, USA) and then transferred to a nitrocellμlose membrane (Bio-rad, California, USA). The membrane-transferred proteins were blocked with 5% skim milk (Bio-rad, Seoul, South Korea) for 1 hour, and then anti-PARP (Cell signaling Technology, Massachusetts, USA) and anti-Caspase-3 (Cell signaling Technology, Massachusetts, USA) antibodies were diluted in 5% skim milk, respectively, and the membrane was placed in a solution and reacted at 4°C for 16 hours. After washing three times with 1X TBST for 10 minutes, the secondary antibody, mouse anti-rabbit IgG HRP (Santa Cruz Biotechnology, Texas, USA), was diluted in 5% skim milk and reacted for 2 hours at room temperature, and washed three times with 1X TBST for 10 minutes. Luminol / enhancer solution (Cyanagen, Bologna, Italy) and Peroxide solution (Cyanagen, Bologna, Italy) were mixed in a 1:1 ratio and applied to the membrane, reacted for 1 minute at room temperature, and protein expression was confirmed using a fusion solo X (Vilber, Eberhardzell, Germany) device (Fig. 4 (B)). This means that the PENK gene can effectively inhibit the proliferation of bladder cancer cells by inducing apoptosis compared to the control gene, and can be used as a bladder cancer treatment. Example 4. Confirmation of PENK's cell proliferation and colony formation inhibition functions. 4-1. Confirmation of cell proliferation inhibition function (cell growth inhibition test) Bladder cancer T24 cell line was seeded at 0.05 x 10 in a 24-well plate (SPL, Pocheon, Korea). 6 After spreading the cells per well, they were cultured in a 37℃ incubator under 5% CO2 conditions for 24 hours. mRNA synthesized by the IVT method and Lipofectamine Messenger MAX were mixed (1:1.5, w:v) and transfected into T24 cells, and cultured in a 37℃ incubator under 5% CO2 conditions for 24 hours. After processing using the Cell counting kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions, the cells were cultured in a CO2 incubator for 1 hour, and the absorbance (450 nm) of living cells was measured using mobi (Microdigital, Seoul, Korea). As a result of checking the absorbance of the cells, it was confirmed that the proliferation of cells transfected with PENK mRNA was reduced by approximately 37% compared to cells transfected with control mRNA (Fig. 5). This means that the PENK gene can effectively inhibit the proliferation of bladder cancer cells and can be used as a bladder cancer treatment. 4-2. Confirmation of cell colony formation inhibition function (colony formation assay) Bladder cancer T24 cell line was seeded at 0.8 x 10 in a 60Ø plate (SPL, Pocheon, Korea). 6After spreading the cells per well, they were cultured for 24 hours in a 37°C incubator under 5% CO2 conditions. The mRNA synthesized by the IVT method and Lipofectamine Messenger MAX were mixed (1:1.5, w:v) and transfected into the T24 cell line. After culturing for 24 hours in a 37°C incubator under 5% CO2 conditions, the cells were harvested. The harvested cells were seeded in a 6-well plate at a density of 3 x 10 3 After seeding cells per well, they were cultured in a 37℃ incubator under 5% CO2 conditions for 7 days. After confirming colony formation, the medium in each well was removed and fixed by treating with 100% methanol at room temperature for approximately 20 minutes. After cell fixation, they were washed with distilled water, stained with 0.5% Crystal violet, washed with distilled water, and checked the results. As a result, it was confirmed that cell cluster formation of the PENK gene was reduced by approximately 37% compared to the control gene (Fig. 6). This means that the PENK gene can effectively suppress colony formation of bladder cancer cells and can be used as a bladder cancer treatment. Example 5. Confirmation of PENK's cell migration and invasion inhibition function. 5-1. Wound healing assay Bladder cancer T24 cell line was seeded at 0.05 x 10 in a 24-well plate (SPL, Pocheon, Korea). 6After spreading the cells per well, they were cultured in a 37℃ incubator under 5% CO2 conditions for 24 hours. mRNA synthesized by the IVT method and Lipofectamine Messenger MAX were mixed (1:1.5, w:v) and transfected into T24 cells, and cultured in a 37℃ incubator under 5% CO2 conditions for 24 hours. Afterwards, a ScarTM Scratcher (SPL, Pocheon, Korea) was used to make a wound of a certain width and washed with PBS. After culturing for an additional 24 hours, the distance of cell migration in the wound area was measured using Image J software from photographs taken before and after culturing. As a result of measuring the migration distance, the wound healing ability of the PENK gene was reduced by approximately 40% in 24 hours compared to the control gene, indicating that the cell migration and wound healing ability of the PENK gene were reduced (Fig. 7). This means that the PENK gene can be used as a bladder cancer treatment by effectively inhibiting the proliferation of bladder cancer cells by significantly reducing cell migration compared to the control gene. 5-2. Transwell invasion assay Bladder cancer T24 cell line was seeded at 0.05 x 10 in a 24-well plate (SPL, Pocheon, Korea). 6 After spreading the cells per well, they were cultured in a 37°C incubator under 5% CO2 conditions for 24 hours. The mRNA synthesized by the IVT method and Lipofectamine Messenger MAX were mixed (1:1.5, w:v) and transfected into the T24 cell line, and then cultured in a 37°C incubator under 5% CO2 conditions for 24 hours, and the cells were harvested. First, 1x10 4Cells were added at a concentration of 100 cells per well, and RPMI supplemented with serum was added to the bottom. The cells were cultured for 24 hours in a 37℃ incubator under 5% CO2 conditions. After fixation and staining using the Deep Quick Kit (sysmex, Kobe, Japan), cells were counted under a microscope. Compared to the Control gene, the degree of Transwell invasion of the PENK gene was reduced by approximately 50% (Fig. 8). This suggests that the PENK gene regulates the motility of bladder cancer cells, significantly reducing their migration and invasion capabilities, and can be used as a bladder cancer treatment. According to the present invention, a composition for preventing or treating bladder cancer can be provided that inhibits bladder cancer cell growth, induces apoptosis, inhibits cancer cell mobility, and suppresses invasion effects. While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents. Electronic file attached.

Claims

1. A composition for preventing or treating bladder cancer comprising a nucleic acid encoding PENK (Proenkephalin) protein.

2. A composition according to claim 1, characterized in that the nucleic acid comprises a sequence represented by sequence number 1 or a sequence having 90% or more homology therewith.

3. A composition according to claim 1, wherein the nucleic acid is DNA or RNA, and if the nucleic acid is RNA, T in the sequence is U.

4. In the first paragraph, the nucleic acid comprises a 5' untranslated region (UTR) located upstream of the nucleic acid encoding the PENK protein; and A composition comprising a 3' untranslated region (UTR) located downstream of the coding region, wherein the 5' untranslated region (UTR) is included in a genetic construct comprising a leader sequence of a coronavirus.

5. A composition according to claim 4, characterized in that the 5' untranslated region (UTR) comprises a sequence represented by SEQ ID NO:

2.

6. A composition according to claim 4, characterized in that the 3' untranslated region (UTR) comprises a sequence represented by SEQ ID NO:

3.

7. A composition according to claim 4, characterized in that the genetic structure further comprises a promoter and / or poly(A) sequence.

8. A composition characterized in that it contains a genetic construct comprising a sequence represented by sequence number 4 in the first paragraph.

9. A composition for preventing or treating bladder cancer, comprising a nucleic acid encoding PENK (Proenkephalin) protein and a carrier.

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